filaminbplaysakeyroleinvascularendothelialgrowth factor ... · westernblotting...

Filamin B Plays a Key Role in Vascular Endothelial GrowthFactor-induced Endothelial Cell Motility through ItsInteraction with Rac-1 and Vav-2*□S

Received for publication, September 7, 2009, and in revised form, January 25, 2010 Published, JBC Papers in Press, January 28, 2010, DOI 10.1074/jbc.M109.062984

Beatriz del Valle-Perez‡§, Vanesa Gabriela Martínez§, Cristina Lacasa-Salavert‡§, Agnes Figueras§,Sandor S. Shapiro¶†, Toshiro Takafuta�, Oriol Casanovas§, Gabriel Capella§, Francesc Ventura‡,and Francesc Vinals‡§1

From the ‡Unitat de Bioquímica i Biologia Molecular, Departament de Ciencies Fisiologiques II, Universitat de Barcelona-IDIBELLand the §Laboratori de Recerca Translacional, Institut Catala d’Oncologia-IDIBELL, L’Hospitalet de Llobregat,E-08907 Barcelona, Spain, the ¶Department of Molecular Physiolgy and Biophysics, Jefferson Medical College of ThomasJefferson University, Philadelphia, Pennsylvania 19107, and �Nishi-Kobe Medical Center, Kobe, Hyogo 651 2273, Japan

Actin-binding proteins filamin A (FLNA) and B (FLNB) areexpressed in endothelial cells and play an essential role duringvascular development. In order to investigate their role in adultendothelial cell function,we initially confirmed their expressionpattern in different adult mouse tissues and cultured cell linesand found that FLNB expression is concentrated mainly inendothelial cells, whereas FLNA is more ubiquitously ex-pressed. Functionally, small interfering RNA knockdown ofendogenous FLNB in human umbilical vein endothelial cellsinhibited vascular endothelial growth factor (VEGF)-inducedin vitro angiogenesis by decreasing endothelial cell migrationcapacity, whereas FLNAablation did not alter these parameters.Moreover, FLNB-depleted cells increased their substrate adhe-sion with more focal adhesions. The molecular mechanismunderlying this effect implicatesmodulationof smallGTP-bindingproteinRac-1 localizationandactivity,withalteredactivationof itsdownstream effectors p21 protein Cdc42/Rac-activated kinase(PAK)-4/5/6 and its activating guanine nucleotide exchange factorVav-2. Moreover, our results suggest the existence of a signalingcomplex, including FLNB, Rac-1, and Vav-2, under basal condi-tions that would further interact with VEGFR2 and integrin �v�5after VEGF stimulation. In conclusion, our results reveal a crucialrole for FLNB in endothelial cell migration and in the angiogenicprocess in adult endothelial cells.

Angiogenesis, or the formation of new blood vessels frompreexisting vasculature, is an essential part of many physiolog-

ical processes, including wound healing and the female men-strual cycle. However, stimulated blood vessel growth is alsofound in many disease states, such as tumor growth, diabeticretinopathy, and arthritis (1–3). Among the best studied induc-ers of angiogenesis areVEGF,2 fibroblast growth factor 2, trans-forming growth factor-�, and angiopoietins.Angiogenesis is a complex process with several stages,

including disruption of the intercellular junctions, extracellularmatrix remodeling, sprouting, endothelial cell proliferation,and migration. The alterations that allow this process arereversible because once at their destination, endothelial cellsstop dividing, regenerate the extracellular matrix, and reestab-lish intercellular contacts (3–5). All of these changes requiremajor reorganization of the endothelial cell cytoskeleton, theintegrity of which is essential for the maintenance of cell struc-ture. Furthermore, it is known that remodeling of the actincytoskeleton accompanies alterations in cell shape andmotilityduring processes such as normal organogenesis, oriented nerveand capillary growth, and angiogenesis (6). A host of actin-binding proteins play a role in cytoskeletal organization (6, 7),many of which are also involved in connecting the plasmamembrane and its integral proteins, such as integrins, with theactin cytoskeleton (8). The actin-binding protein family in-cludes talin, vinculin, �-actininin, and the filamins, among oth-ers. Endothelial cells contain high levels of these proteins, pre-sumably in order to maintain a structured actin cytoskeletonand the capacity to respond to signals that will affect motilityand cell structure. Factors that induce motility of endothelialcells, such as angiogenic cytokines (VEGFand fibroblast growthfactor 2), affect the actin cytoskeleton and also actin-bindingproteins (9–11).Filamins bind actin through their N-terminal actin-binding

domain (ABD), whereas the C-terminal 24th repeat is essentialfor filamin dimerization (12–14). The ABD is followed by aseries of 24 repeated sequences of �100 amino acids that func-

* This work was supported, in whole or in part, by National Institutes of HealthGrant HL-072439 (to S. S. S. and T. T.). This work was also supported byMinisterio de Ciencia y Tecnología Grants SAF2004-01350, SAF2007-60955, and RTICC RD2006-0092 (to F. Vinals) and BFU2008-02010 to(F. Ventura), Generalitat de Catalunya Grants 2005SGR727 and2009SGR283 (to F. Vinals), and a grant from the National HemophiliaFoundation, Delaware Valley Chapter (to S. S. S.).

This paper is dedicated to the memory of Dr. Sandor S. Shapiro, who passedon in July 2007 while experimental work was in progress.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4 and Videos 1 and 2.

† Deceased July 21, 2007.1 To whom correspondence should be addressed: Laboratori de Recerca

Translacional, Institut Catala d’Oncologia-IDIBELL, Gran Via S/N KM 2,7,E-08907 L’Hospitalet de Llobregat, Barcelona, Spain. Tel.: 34-93-260-7344;Fax: 34-93-260-7466; E-mail: [email protected].

2 The abbreviations used are: VEGF, vascular endothelial growth factor;HUVEC, human umbilical vein endothelial cell(s); FCS, fetal calf serum; GFP,green fluorescent protein; siRNA, small interfering RNA; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; GST, glutathioneS-transferase; GAP, GTPase-activating protein; FLNA and -B, filamin A andB, respectively.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10748 –10760, April 2, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

10748 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 14 • APRIL 2, 2010

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tion as domains of protein-protein interaction; so far, morethan 30 different proteins have been described as interactingwith filamins. The function of filamins seems to be dual: first,helping to form a three-dimensional network of actin throughtheir actin-binding domain; and second, acting as scaffoldingproteins offering docking sites for cytoplasmic proteins, mem-brane receptors, integrins, etc. Thus, filamins connect plasmamembrane with the intracellular cytoskeleton and are involvedin changes in cell shape (12–14). Three filamins have beendescribed: filamin A, or ABP280, is the most abundant and iswidely expressed in different tissues; filamin B is also broadlyexpressed; and filamin C is mainly expressed in skeletal muscle(12–14). High levels of filamin A and filamin B have previouslybeen detected in vessels (15–17) and in endothelial cells in cul-ture (17–19). Knock-out experiments show the functionalimportance of each isoform: filamin A knock-out mice die dur-ing development due to aberrant vascular patterning and car-diac defects (20, 21), whereas filamin B deficiency in micemainly causes skeletal malformations (22–24) and vasculardeficiencies (23).In order to study the role of both filamins A and B in vascular

endothelial cells, we abrogated their expression in humanumbilical vein endothelial cells (HUVEC) in vitro. Using thismodel, we show in this work a key role for filamin B in endo-thelial cellmotility, exerted through its function as a scaffoldingprotein connecting VEGFR2, Vav-2, and Rac-1 proteins.

EXPERIMENTAL PROCEDURES

Materials

VEGFwas fromCalbiochem. Cell culturemedia, fetal bovineserum, glutamine, and antibiotics were obtained from Invitro-gen or Biowhittaker. Other reagents were of analytical ormolecular biology grade and were purchased from Sigma orRoche Applied Science.

Cell Culture and Transfections

Murine lung capillary endothelial cells (cell line 1G11) wereobtained from Alberto Mantovani and Annunciata Vecchi(Instituto Ricerche Farmacologiche Mario Negri, Milan, Italy)(25). They were cultured in Dulbecco’s modified Eagle’smedium, containing 20% FCS, 50 units/ml penicillin, 50 �g/mlstreptomycin sulfate, 25 �g/ml endothelial cell growth supple-ment (BD Biosciences), 100 �g/ml heparin (Sigma), 1% non-essential amino acids, and 2 mM sodium pyruvate.

HUVEC were obtained from Advancell (Barcelona, Spain)and were cultured in M199 (Biowhittaker) supplemented with20% FCS, 50 units/ml penicillin, 50�g/ml streptomycin sulfate,25 �g/ml endothelial cell growth supplement, 100 �g/ml hep-arin, 2 mM sodium pyruvate, 1 mM HEPES, pH 7.4. HUVECwere transiently transfected using Lipofectamine 2000. Plas-mids used were GFP, GFP-Rac-1-Valine12 (a constitutivelyactive formof Rac-1), andGFP-Rac-1-M7 (a dominant negativeform of Rac-1), the last two a generous gift from Dr. Alan Hall(Memorial Sloan-Kettering Cancer Center, New York).Human siRNAs for FLNB and FLNAwere constructed using

the SilencerTM siRNA construction kit (Ambion), according tothe manufacturer’s instructions. Three independent sequencesfor human filaminB and two independent sequences for human

filamin A were designed with free Whitehead Institute soft-ware (available on the World Wide Web). siRNA-FLNB1corresponded to 5�-AAGCTCCCTTAAAGATATTTG-3�(nucleotides 2031–2051 of filamin B); siRNA-FLNB2 corre-sponded to 5�-AAGGTCCTTCCCACATATGAT-3� (nucle-otides 4529–4549); and siRNA-FLNB3 corresponded to5�-AACACCTGAAGGGTACAAAGT-3� (corresponding tonucleotides 7299–7319). siRNA-FLNA1 corresponded to 5�-AAGATGTCCTGCATGGATAAC-3� (nucleotides 4637–4389 of filamin A). siRNA-FLNA2 corresponded to 5�-AAG-ACCACCTACTTTGAGATC-3� (nucleotides 1370–1392).No similar sequences (withmore than 17 identical nucleotides)were found in BLAST. Irrelevant siRNA corresponded to thenegative universal control RNA interference (Invitrogen). ForsiRNA experiments, HUVECwere seeded in 6-well plates. Fourhours before transfection, medium was changed to Opti-MEMI without antibiotics and supplemented with 20% FCS. siRNAswere then transfected with Lipofectamine 2000. The totalamount of siRNA (final concentration 62.5 nM) was diluted inOpti-MEM I; in a different tube, Lipofectamine 2000 was alsodiluted in Opti-MEM I. After a 5-min incubation at room tem-perature, the transfection reagent mix was added to the siRNAand was further incubated for 20 min at room temperature.Cells were incubatedwith the reactionmix for 4 h. At the end ofthe incubation, cells werewashed twicewithOpti-MEM Iwith-out antibiotics and supplemented with 10% FCS. Media werereplaced with 2 ml of complete Medium 199.

Tubulogenesis Assays

For HUVEC, 2 � 105 cells were seeded in triplicate in a24-well plate coated with 300 �l of rat tail type-I collagen (1.2mg/ml) in Dulbecco’s modified Eagle’s medium and 20% FCSand allowed to attach for at least 2 h. Following this, cells wereoverlaid with an additional 300 �l of collagen. Once the colla-gen had polymerized, cells were fed with M199 medium, 1%FCS, 100 �g/ml heparin, 150 �g/ml endothelial cell growthsupplement, and 40 ng/ml VEGF. Following completion of net-work formation (24 h), 3 images/well were captured on a Leicainverted phase-contrast microscope DMIRBE equipped withdigital capture software, and tubulogenesis wasmeasured usingImageJ software.

Migration Assays

Migrationwasmeasured using a Boyden chamber. Transwellinserts (8-�m pore size; Costar) were coated with fibronectin(25 �g/ml) overnight at 4 °C. Transwells were washed withphosphate-buffered saline (PBS) and blocked with PBS con-taining 5% bovine serum albumin for 3 h at 37 °C. Transwellswere rinsed with PBS and dried for 3 h in a laminar flow cabin.HUVEC were seeded into the transwell (200,000/well), and

after 1 h at 37 °C,migrationwas stimulated by the addition of 20ng/ml VEGF. After 16 h, transwells were washed, and cells werefixed with 3% paraformaldehyde for 30 min. After four washeswith PBS, they were stained with 5 �g/ml propidium iodide for5min and rinsed four timeswith PBS, and the upper surfacewaswiped to remove the non-migrating cells. Imageswere capturedon a Leica inverted immunofluorescence microscope, and themigrating cells were counted.

Role of Filamin B in Endothelial Cell Motility

APRIL 2, 2010 • VOLUME 285 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 10749

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Western Blotting

Cells were washed twice in cold PBS and lysed for 15 min at4 °C in Triton X-100 lysis buffer (50 mM Tris-HCl, 100 mM

NaCl, 50 mM NaF, 5 mM EDTA, 40 mM �-glycerophosphate,200 �M sodium orthovanadate, 100 �M phenylmethylsulfonylfluoride, 1 �M pepstatin A, 1 �g/ml leupeptin, 4 �g/ml aproti-nin, 1%TritonX-100, pH 7.5).Western blotting was performedas described (26). The blots were incubated with polyclonalrabbit anti-filamin B antibody (17), monoclonal anti-filamin Aantibody (Chemicon), polyclonal anti-PARP antibody (CellSignaling), monoclonal anti-VEGFR2 antibody (Santa CruzBiotechnology, Inc., Santa Cruz, CA), polyclonal anti-VEGFR1 antibody (Santa Cruz Biotechnology, Inc.), mono-clonal anti-RhoA antibody (Santa Cruz Biotechnology, Inc.),polyclonal anti-phospho-VEGFR2 (Tyr1175; Cell Signaling),polyclonal anti-phospho-Src antibody (Tyr527; Cell Signal-ing), polyclonal anti-Src (Cell Signaling), monoclonal anti-Rac-1 antibody (Upstate), polyclonal anti-phospho-PAK-4/5/6 antibody (Cell Signaling), polyclonal anti-PAK-4antibody (Cell Signaling), polyclonal anti-phospho-Vav-2antibody (Santa Cruz Biotechnology, Inc.), polyclonal anti-Vav-2 antibody (Santa Cruz Biotechnology, Inc.), mono-clonal anti-phospho-ERK antibody (Sigma), polyclonal anti-ERK1/2 antibody (27), monoclonal anti-�-tubulin antibody(Sigma), or monoclonal anti-�-actin antibody (Sigma) inblocking solution overnight at 4 °C.

Immunoprecipitation

Quiescent HUVEC stimulated or not with 10 ng/ml VEGFfor 5minwere lysed with octyl glucopyranoside buffer (100mM

octyl glucopyranoside, 2mMCaCl2, 2mMMgCl2, 40mM �-glyc-erophosphate, 200 �M sodium orthovanadate, 100 �M phenyl-methylsulfonyl fluoride, 1 �M pepstatin A, 1 �g/ml leupeptin, 4�g/ml aprotinin in PBS, pH 7.5) for 15 min at 4 °C. Insolublematerial was removed by centrifugation at 13,000 � g for 20min at 4 °C. Protein lysates (750 �g) were precleared with 30 �lof protein A/protein G-Sepharose beads (Amersham Bio-sciences) for 2 h at 4 °C, centrifuged at 13,000 � g for 10 min at4 °C, and incubated overnight with monoclonal anti-VEGFR2antibody (Santa Cruz Biotechnology, Inc.), polyclonal anti-VEGFR1 antibody (Santa Cruz), monoclonal anti-Rac-1 anti-body (Upstate), monoclonal anti-RhoA antibody (Santa Cruz),polyclonal anti-Vav-2 antibody (Santa Cruz), or monoclonalanti-�v�5 (Merck Farma y Química, Barcelona, Spain). Sam-ples were incubated with 30 �l of protein A/protein G-Sepha-rose beads (Amersham Biosciences) for an additional 4 h at4 °C. Precipitates were washed four times with Triton X-100lysis buffer (50 mM NaF, 40 mM �-glycerophosphate, 200 �M

sodium orthovanadate, 100 �M phenylmethylsulfonyl fluoride,1 �M pepstatin A, 1 �g/ml leupeptin, 4 �g/ml aprotinin, 0.1%Triton X-100 in PBS, pH 7.5). The final pellet was resuspendedin 50 �l of Laemmli sample buffer (28), followed by proteinseparation on an SDS-7.5% polyacrylamide gel and Westernblotting as described before using the appropriate antibodies.

GST-Rac-1 Pull-down Assay

Quiescent HUVEC stimulated or not with 10 ng/ml VEGFfor 5minwere lysedwith TritonX-100 lysis buffer (50mMNaF,

40 mM �-glycerophosphate, 200 �M sodium orthovanadate,100 �M phenylmethylsulfonyl fluoride, 1 �M pepstatin A, 1�g/ml leupeptin, 4 �g/ml aprotinin, 0.1% Triton X-100 in PBS,pH 7.5) for 15 min at 4 °C. Insoluble material was removed bycentrifugation at 13,000 � g for 20 min at 4 °C. Protein lysates(750 �g) were incubated with equal amounts of GST or GST-Rac-1 (a generous gift from Dr. Mireia Dunach, UniversitatAutonoma de Barcelona) overnight at 4 °C. After this time, 15�l of glutathione-Sepharose beads (Amersham Biosciences)were added for an additional 4 h at 4 °C. Beads were washedfour times with Triton X-100 lysis buffer. The final pellet wasresuspended in 30 �l of Laemmli sample buffer, followed byWestern blotting as described before with a polyclonal rabbitanti-filamin B antibody or a monoclonal mouse anti-GST.

Rac G-LISA

For measuring Rac-1-GTP levels, HUVEC cells wereserum-starved overnight. Rac-1-GTP was detected using thecolorimetric G-LISA Rac-1/2/3 activation assay (Cytoskele-ton, Denver, CO). Briefly, cells were lysed according to themanufacturer’s protocol. Total protein was measured,appropriately diluted in binding buffer, and incubated on96-well plates that contained a Rac-GTP-binding proteinlinked to the bottom of each well. The bound Rac-GTP isdetected with a Rac-specific primary antibody and a horse-radish peroxidase-conjugated secondary antibody. The sig-nal produced by the horseradish peroxidase detection rea-gent is proportional to the amount of Rac-GTP and can bedetected by measuring absorbance at 595 nm. Appropriatecontrols were carried out (positive control was Rac-1 controlprotein, and negative control was lysis buffer alone).

Northern Blotting

Total RNA from cells was extracted using the phenol/chlo-roformmethod, andNorthern blotting using 20�g of RNAwasperformed as described (26). Blots were hybridized to mousefilamin B cDNA, to a fragment of the 3�-end of the humanfilamin A sequence (a generous gift from Dr. Saret (NationalInstitutes of Health)) or to rat glyceraldehyde-3-phosphatedehydrogenase cDNA labeled with [�-32P]dCTP (AmershamBiosciences).

Immunofluorescence Studies

Two-dimensional Cell Immunofluorescence—Cells were cul-tured on glass coverslips for 24 h, rinsed three times with PBS,and fixed in 3%paraformaldehyde for 30min. After fourwasheswith PBS, theywere permeabilizedwithPBS, 0.2%TritonX-100for 5 min, rinsed four times with PBS and blocked for 30 min atroom temperature in PBS containing 2% bovine serum albu-min. Coverslips were incubated with polyclonal rabbit anti-fil-amin B antibody (17), monoclonal anti-filamin A antibody(Chemicon), monoclonal anti-Rac-1 antibody (Upstate), ormonoclonal anti-vinculin antibody (Sigma), followed by 5�g/ml Alexa 488 anti-rabbit or Alexa 568 anti-mouse (Molec-ular Probes) for 1 h at room temperature. To visualize F-actin,coverslips were incubated with red-phalloidin or 633-phalloi-din (colored blue) 1 �g/ml (Sigma) for 1 h. Coverslips weremounted using Gel Mount (Biomedia), and fluorescence was



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viewed with a Leica confocal microscope (LEICA spectral con-focal TCS-SL, Serveis Científico Tecnics de la Universitat deBarcelona).Tissue Immunofluorescence—Cryopreserved sections of

mouse tissue were analyzed for filamin B (polyclonal rabbitantibody) (17), filaminA (monoclonalmouse antibody, Chemi-con), PECAM/CD31 (monoclonal rat antibody, BD Pharmin-gen), vonWillebrand factor (polyclonal rabbit antibody, Dako),and �-smoothmuscle actin (polyclonal rabbit antibody, LabVi-sion). Primary antibodies were incubated overnight at 4 °C(1:50). Nuclei were stained with TO-PRO-3 iodide 642/661 (0.2�M; Molecular Probes). Appropriate secondary antibodiesbound to the appropriate fluorochrome (Molecular Probes)were used. Negative controls (lacking primary antibody butcontaining the secondary antibody) confirmed the specificity ofthe staining.Tissue Immunohistochemistry—Sections from representa-

tive paraffin blocks of mouse tissues were deparaffined, rehy-drated, and blocked for endogenous peroxidase activity using3% H2O2. Antigen was retrieved with heat in sodium citratebuffer (8.2 mM trisodium citrate dihydrate and 1.98 mM citricacid, pH 6.0) or digestion with 0.1% trypsin in 1� PBS buffer.Primary polyclonal rabbit anti-filamin B antibody (1:10) wasincubated for 16 h. Appropriate secondary antibodies wereused. After development with diaminobenzidine, substrate wascounterstained with Harris’s hematoxylin. No staining wasobserved with negative control samples (absence of primaryantibody or incubation with an irrelevant antibody or IgG).

Time Lapse Imaging

After transfection with siRNAs and GFP (to visualize trans-fected cells), HUVEC were starved for 12 h and then culturedfor a further 12 h in the presence of 20 ng/ml VEGF. Duringthese 12 h, GFP-positive cells were imaged by phase-contrastevery 5 min on a LEICA spectral confocal TCS-SL with a �20objective. Images were analyzed using ImageJ software. Weperformed analysis of the track of 10 different cells from twoindependent experiments.

Cell Cycle Analysis

After transfection with siRNAs, HUVEC were starved for12 h and then cultured for a further 12 h in basal medium orcompletemedium. Cells were then trypsinized and pooled withfloating cells in the culture medium, washed, and fixed in 70%ethanol in PBS; they were kept in ethanol at �20 °C for at least24 h. For staining, cells were washed to remove ethanol andresuspended in PBS with 100 �g/ml RNase and 40 �g/ml pro-pidium iodide and then incubated at 37 °C for at least 30 minbefore flow cytometry analysis.

Statistical Analyses

Statistical significance of differences between the effects ofunrelated siRNA and siRNAs against filamins were determinedusing Student’s t test. In all experiments, differences were con-sidered statistically significant when p was �0.05.

RESULTS

To confirm the vascular expression of filamins A and B, weperformed immunofluorescence and immunohistochemistry

on sections of adult mouse tissues (lung, kidney, adipose tissue,and testis). Using immunofluorescence on cryopreserved tissuesections, we detected low filamin B expression in parenchymalcells of lung (data not shown) and testis (supplemental Fig. S1A)and high expression levels in blood vessels, with a pattern verysimilar to that of PECAM/CD31, a classical endothelial cellmarker. In contrast, we found high expression of filamin A inthe parenchyma and in the different cell types that form thevessels, endothelial and mural cells, co-localizing with vonWillebrand factor (a marker of endothelial cells) and with�-smooth muscle actin (a mural cell marker) (supplementalFig. S1A). Both types of filamins, A and B, were expressed inendothelial cells. Using immunohistochemistry, we detectedfilamin B expression in vessels of lung, kidney, and adipose tis-sue (supplemental Fig. S1B), with lower levels observed inparenchymal cells. Taken together, these results indicate thatthe expression pattern of filamins in vessels is isoform-specific,with filamin A expressed at similar levels in the different celltypes, whereas filamin B expressionwas concentratedmainly inendothelial cells.Next, we analyzed filaminA andB expression in different cell

lines and primary cultures by performing Northern blottingexperiments using specific probes for filamin A and B andWestern blotting experiments using specific antibodies. Usingthese reagents, we detected high levels of filamin B (bothmRNA and protein) in primary HUVEC cultures (Fig. 1, A andB) and other endothelial cell lines, such as 1G11 (a murine lungcapillary endothelial cell line) (25) and H5V (a mouse heartendothelial cell line transformed by polyomamiddle T antigen)(29) (supplemental Fig. S1C) compared with other cell lines.We also detected filamin B protein expression in HeLa, C2C12,and HT29 (human colorectal adenocarcinoma) cells, butexpression levels in these cell lines weremuch lower than thoseof endothelial cells (Fig. 1,A and B, and supplemental Fig. S1C).In contrast, filamin A was expressed at roughly the same levelsin the different cell types studied, including endothelial cells(Fig. 1, A and B), further confirming the high levels of endothe-lial FLNB expression and a broader expression of FLNA in var-ious cell types.In order to study the role of filamin A and B in vascular

endothelial cells, we chose primary cultures ofHUVECbecausethey are much more representative of normal endothelial cellphysiology as compared with the other cell lines. We sought toelucidate the functional importance of each filamin isoform inHUVEC tubule formation by an in vitro angiogenesis assay. Todo so, we generated three independent siRNAs against threedifferent sequences of human filaminBmRNA (siRNA-FLNB1,-2, and -3) and two siRNAs for filamin A (siRNA-FLNA1 and-2). We transfected HUVEC with these siRNAs and evaluatedtheir effects on filamin A and filamin B expression by Westernblotting. We found that siRNA-FLNB1 decreased filamin Bexpression by 40–60%, whereas siRNA-FLNB2 and siRNA-FLNB3 were the most effective with a decrease in filamin Blevels of 70–80%; these siRNAs showed no effect on filamin Aexpression (Fig. 1C). The siRNAs against filamin A caused adramatic decrease in filamin A protein levels, an effect thatappeared to be compensated by an increase in filamin B expres-sion (Fig. 1C). To evaluate the role of filamins in in vitro angio-



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genesis, we transfected HUVEC with these siRNAs and, 24 hlater, cultured the cells for an additional 24 h in type-I collagengels in the absence or presence of the most important proan-giogenic factor, VEGF (Fig. 2A). When cells were analyzed fortheir capacity to form tubular structures in type-I collagen gels,we discovered that siRNA-FLNBs blocked VEGF-inducedtubulogenesis. In contrast, when we evaluated the effect of thesiRNAs designed against filamin A on in vitro angiogenesis incollagen gels, we found that they did not affect endothelial cellreorganization after VEGF treatment (Fig. 2A).Next, we searched for a mode of action for filamin B that

could explain the results obtained in the in vitro assay. Trans-fection of HUVEC with the different siRNAs did not affectendothelial cell apoptosis or proliferation (supplemental Fig.S2,A and B). This effect was also confirmed by the lack of effectof filamin B depletion on a typical proliferation signaling path-way (ERK stimulation; see Fig. 5B). We next studied a possible

role for this protein in endothelialcell migration. Given the role of fil-amins as actin-binding proteins, wehypothesized that filamin B couldinfluence this process. Thus, wemeasured migration in Boydenchambers of HUVEC transfectedwith control, filamin A, or filamin BsiRNAs and stimulated or not withVEGF. As observed in Fig. 2B, abla-tion of filamin B decreased basalHUVEC migration (55 and 78%decrease for siRNA-FLNB2 and-FLNB3, respectively) and abol-ished VEGF-stimulated cell migra-tion. However, down-regulation offilamin A caused only a 22%decrease in basal migration and didnot affect VEGF-stimulated migra-tion. These data confirm the criticalrole of filamin B in endothelial cellmigration and, consequently, inangiogenesis, whereas filamin Aappears to play a secondary and lessimportant role in this process.In order to identify the mecha-

nisms through which filamin Baffects cell migration, we first evalu-ated the effect of VEGF stimulationon cytoskeletal organization in con-trol or filamin B-depleted HUVEC.Cells depleted of filamin B pre-sented a flat aspect and a larger sizeas compared with control cells (Fig.3A). After VEGF stimulation, con-trol and filamin B-depleted cellsincreased stress fiber formation.However, cells with low levels of fil-amin B presented multiple cellularprotrusions with polymerized F-ac-tin in different directions (Fig. 3A).

In order to confirm these results, we performed time lapsemicroscopy. Cells were treated with VEGF and left to migrate;images were then captured for the following 12 h. We com-pared the random migration of HUVEC treated with siRNA-FLNB or control siRNA. Using this approach, we observed thatcontrol cells presented a normal migration pattern, defining amigration front and retracting the rear of the cell in order toadvance (supplemental Video 1). In contrast, filamin B ablationdecreased the track and speed of migration (33.8 � 7.3 �m in12 h in control cells, 15.6� 4.6�m in 12 h in filamin B-depletedcells; p � 0.001), and, according to the previous results, cellspresented different migration fronts and could not advanceproperly due to problems in retraction. Many cells ended upgeneratingmultiple protrusions in different directions (supple-mental Video 2).Our results so far suggested decreasedmigration of endothe-

lial cells due to defects in the establishment of new adhesions or

FIGURE 1. Filamin A- and B-specific knockdown by siRNA transfection in endothelial cells. A, Northernblotting analysis. HUVEC, HT29, and HEK-293 (human embryonic kidney) cells are shown. Filamin B, filamin A,and glyceraldehyde-3-phosphate dehydrogenase (as a loading control) were detected using specific probes. Arepresentative Northern blot of three different experiments is shown. B, Western blotting analysis. HUVEC,MCF7 (human breast cancer), HeLa (human cervical adenocarcinoma), HEK-293, HT29, and COS (green monkeykidney) cells are shown. Filamin B, filamin A, and actin (as a loading control) were analyzed by immunoblotting.A representative blot of three different experiments is shown. C, HUVEC were transiently transfected with acontrol unrelated siRNA (Unr), siRNA-FLNB1 (1), siRNA-FLNB2 (2), siRNA-FLNB3 (3), siRNA-FLNA1 (1), or siRNA-FLNA2 (2), as described under “Experimental Procedures.” After 48 h, cells were lysed as described, and filaminB (FLNB), filamin A (FLNA), or �-actin (as a loading control) were detected by immunoblotting. Two (for siRNA-FLNB) or three (for siRNA-FLNA) independent experiments are shown for each siRNA.



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in retraction. To evaluate the effect of filamin B on adhesion tothe extracellular matrix, we performed vinculin immuno-staining using a specific antibody. As is shown in Fig. 3B, con-trol HUVEC presented low levels of focal adhesions and con-tacts in the basal state, which increased after VEGF stimulation.Filamin B ablation caused a dramatic increase in focal adhe-sions and contacts in the basal state; the number of focal adhe-sions did not experience a further increase after VEGF stimu-lation. In contrast, siRNA-mediated filamin A knockdown did

not affect the formation of adhesion contacts in basal or VEGF-stimulated conditions.Different signaling pathways are activated by VEGF in order

to stimulate endothelial cell migration. Focal adhesion kinase,its substrate paxillin, and the Rho family of GTPases areinvolved in focal adhesion turnover and stress fiber formation(10, 30, 31); phosphatidylinositol 3-kinase, ROCK (Rho-associ-ated coiled-coil-containing protein kinase), p38 MAPK, MAP-KAPK-2, andLIMK (LIMdomain kinase 1) are involved in actin

FIGURE 2. Filamin B is essential for VEGF-induced in vitro angiogenesis and endothelial cell migration. A, HUVEC were transiently transfected with anunrelated control siRNA, siRNA-FLNA1 and -FLNA2, or siRNA-FLNB2 and -FLNB3, as described. After 24 h, cells were trypsinized and immersed in collagen gelsin the presence (�VEGF) or absence (Basal) of 40 ng/ml VEGF for an additional 24 h. Four independent images of each experiment were captured on a Leicainverted phase-contrast microscope (�10) to evaluate the tubule formation using ImageJ software (measurement of cell occupied surface). Results are themean � S.E. from three independent experiments. VEGF significantly stimulated tubular structures in control unrelated siRNA (Unr)- and in siRNA-FLNA1- andsiRNA-FLNA2-treated cells (see arrows, p � 0.01, t test) without a significant effect on siRNA-FLNB2 and -FLNB3 cells. B, after transfection with the differentsiRNAs, cells were seeded into a Boyden chamber and left to migrate in the presence or absence of 20 ng/ml VEGF for 16 h. The number of migrating cells inbasal conditions or after VEGF treatment was counted for every siRNA. We show the mean � S.E. from three independent experiments. VEGF significantlystimulated cell migration in control unrelated siRNA- and in siRNA-FLNA-treated cells (p � 0.05, t test) without a significant effect on cells transfected withsiRNA-FLNB2 or -3. siRNA-FLNA1, -B2, and -B3 caused a significant reduction in basal cell migration (p � 0.005, t test).



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reorganization (10, 32, 33); and Src kinases and integrins areinvolved in adhesion and extracellular matrix interaction (34,35). As described previously, filamins interact with a largenumber of intracellular signaling proteins, membrane recep-tors, integrins, etc. that affect different intracellular signal-ing pathways. Taking into account the effects of our siRNAsin cell migration and shape, we hypothesized that filamin Bcould affect VEGF signaling through interaction with VEGFreceptors, integrins, and the Rho family of small GTPaseproteins, so we performed immunoprecipitation experi-ments using antibodies against Rac-1, RhoA, VEGF recep-tors (VEGFR1 and VEGFR2), and �v�5, an integrin that par-ticipates in angiogenesis. After immunoprecipitation, wedetected filamin B by Western blotting. In basal conditions,we only detected interaction of filamin B with Rac-1 (Fig.4A). After VEGF stimulation, filamin B not only maintainedits interaction with Rac-1 but also coprecipitated withVEGFR2 and integrin �v�5 (Fig. 4A). In contrast, filamin Bdid not interact with RhoA or VEGFR1 (supplementalFig. S3). In order to confirm these data, we performed pull-down assays using recombinant glutathione S-transferase

(GST) fused to Rac-1. This construct but not GST specifi-cally interacted with endogenous filamin B from basal orVEGF-stimulated HUVEC (Fig. 4B).Next, we analyzed stimulation by VEGF of some of the

migration signaling pathways in HUVEC cultures. In agree-ment with previous findings, we found stimulation of Rac-1after 1 min of VEGF treatment and a later activation of PAK-4/5/6 (5 min) (supplemental Fig. S4). In contrast, we did notdetect stimulation of LIMK or p38MAPK at the times analyzed(data not shown). Taking into account these results and theobserved interaction between filamin B and Rac-1, we analyzedthe effect of filamin B depletion on the activity of Rac-1. Trans-fection of HUVEC with siRNAs against filamin B increasedbasal Rac-1 activity compared with cells transfected with anunrelated siRNA (Fig. 5A). Consistent with this increase inbasal Rac-1 activity, activation of the Rac-1 substrate PAK-4/5/6 was also increased (Fig. 5B), even in the absence of VEGF,with a sustained stimulation of these proteins observed afterVEGF addition. In contrast, analysis of VEGF-induced ERKactivation in FLNB-depleted cells showed that the decrease infilamin B levels did not affect the capacity of VEGF to stimulate

FIGURE 3. Filamin B knockdown leads to an increase in focal adhesions. A, immunolocalization of F-actin (blue) and filamin A (red) in HUVEC transientlytransfected with an unrelated control siRNA (Unr) or siRNA-FLNB3 as described, in the absence (�VEGF) or presence of 20 ng/ml of VEGF for 30 min (�VEGF).Bar, 40 �m. B, immunolocalization of vinculin (green) and F-actin (red) in HUVEC transfected with an unrelated control siRNA and siRNA-FLNB3, in the absenceor presence of 20 ng/ml VEGF for 30 min. Bar, 10 �m. The graph shows the quantification of focal adhesions in five independent experiments. VEGF significantlystimulated focal adhesions in control unrelated siRNA- and in siRNA-FLNA1-treated cells but not in siRNA-FLNB3 (p � 0.005, t test). siRNA-FLNB3 treatmentsignificantly stimulated focal adhesions compared with basal levels (p � 0.005, t test).



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ERK, which ruled out a nonspecific effect of filamin B depletionon general VEGF signal transduction (Fig. 5B).Taking into account these results, we then evaluatedwhether

filamin B depletion could affect normal Rac-1 function afterVEGF stimulation.We first confirmed that ablation of filaminBby siRNA treatment did notmodify Rac-1 total levels present inHUVEC (Fig. 6A). Next, we examined the effect of filamin B onRac-1 subcellular location. As previously described, after VEGFstimulation of HUVEC, Rac-1 translocated to actin-rich mem-brane structures in the migration front (Fig. 6B). This translo-cation is essential for Rac-1 function (36–38). In contrast, whenfilamin B expression was decreased by siRNA treatment, Rac-1accumulated in the intracellular compartment and was absentfrom the migration front (Fig. 6B). These data indicate thatfilamin B not only can affect Rac-1 activity but also can affect itsintracellular location. Taken together, these results indicatethat loss of filamin B altered normal Rac-1 cycles of activation/inactivation necessary for normal endothelial cell migration.In order to address the importance of Rac-modulated effects

in in vitro tubulogenesis, we overexpressed two different

mutant variants of Rac-1: a consti-tutively active form (GFP-Rac-1-Valine12) and a dominant negativeform of Rac-1 (GFP-Rac-1-M7).Increased activity of Rac-1 by over-expression of the Rac-1-V12 iso-form resulted in HUVEC acquiringa flat phenotype (Fig. 7A), very sim-ilar to the one we observed follow-ing filamin B depletion. When weoverexpressed the dominant nega-tive form of Rac-1, we obtained amixed effect, with some cells pre-senting a flat phenotype and othersappearing unaffected (Fig. 7A).When we assayed cells transfectedwith each isoform in an angiogene-sis assay in vitro, we found thattransfection of either isoformcaused cells to fail to be incorpo-rated into pseudotubules (Fig. 7B),reinforcing the idea that a delicatecontrol of Rac-1 activity is necessaryfor correct tubulogenesis.We next studied whether filamin

B could also affect other proteinsinvolved in the Rac-1 signalingpathway. Rac-1 stimulation byVEGF is mediated by the guaninenucleotide exchange factor Vav-2(39), which is in turn activated byphosphorylated Src. Much as hap-pened with Rac-1, we demonstratedan interaction of filamin B (but notfilamin A) and Vav-2 both in basalconditions and after VEGF stimula-tion (Fig. 8A). Taking into accountthat Rac-1 is overstimulated as a

result of filamin B depletion, we speculated that filamin B siRNAtreatment could also cause Vav-2 overstimulation. Surprisingly,we foundthat incontrastwithourpreviousobservations, filaminBdepletion abolished Vav-2 stimulation by VEGF; levels of activeVav-2 also appeared diminished in basal conditions (Fig. 8B).However, siRNA treatment had no effect on activated Src levels.In all, these results indicate that filamin B depletion significantlyaffects endothelial cell signaling, particularly at the cell adhesionand migration level, blocking Vav-2 stimulation by VEGF andaltering normal Rac-1 activation/inactivation.

DISCUSSION

In the present study, we show that disruption of filamin Bfunction is associated with loss of the proangiogenic effect ofVEGF in three-dimensional collagen gels and inhibition of cellmigration. We propose a key role for filamin B as a scaffoldingprotein coupling Rac-1 to Vav-2 and other extra- and intracel-lular signaling molecules.Our results confirm high levels of filamin B expression in

HUVEC and other endothelial cell types, such as 1G11 and

FIGURE 4. Filamin B interacts with Rac-1 in basal conditions and with Rac-1, VEGFR2, and integrin �v�5in VEGF-stimulated cells. A, cells in basal conditions (Basal) or stimulated with 20 ng/ml VEGF for 5 min(�VEGF) were lysed, and VEGFR2, Rac-1, or �v�5 was immunoprecipitated (IP) with specific antibodies (Ab).Western blotting (WB) for filamin B was performed as described. As a loading control, Western blotting withtotal lysates before immunoprecipitation was also performed (total filamin B). A representative blot of threedifferent experiments is shown. B, pull-down assay using GST or GST-Rac-1 recombinant proteins and lysatesfrom cells in basal conditions (�) or stimulated for 5 min with 20 ng/ml VEGF (�). After pull down, Westernblotting for filamin B or GST was performed as described before. A representative blot of two different exper-iments is shown.



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H5V. In addition, we compared fil-amin A and B expression in vesselsfrom adult mouse tissues. Wereadily detected filamin A expres-sion in vessels, but it was present insmooth muscle cells as well as inendothelial cells; in contrast, filaminB was almost specific to endothelialcells. Co-expression of differenttypes of filamins in the same cell hasbeen described for other cell types(16, 19, 40–42); however, these iso-forms appear to play different func-tions in endothelial cells. Ourresults indicate a key role for filaminB in VEGF-stimulated angiogenesisin vitro in collagen gels,more specif-ically in the regulation of endothe-lial cell motility. Thus, knockdownof filamin B caused a profoundeffect on both endothelial cellmigration and formation of tubularstructures. In contrast, ablation offilamin A expression by siRNAtreatment led only to a slightdecrease in the capacity of endothe-lial cells to migrate. This differentialrole of filamins in the endotheliumcould explain the different vascularphenotypes observed in filamin A-and B-deficient mice. Thus, vesselsfrom filamin A-deficient embryosare present but are disorganized,coarse, and exuberant (20). Endo-thelial cells from these animals pres-ent normal architecture and distri-bution of F-actin, normal filopodia,and membrane protrusions but lowlevels of VE-cadherin and abnormaladherent junctions (20). All of theseresults indicate that knockdown offilamin A causes an alteration inendothelium organization ratherthan a more specific decrease inendothelial cell migration. In con-trast, defects described inmicrovas-culature of filamin B-deficient micemay be the result of altered endo-thelial cell migration (23).Cell migration implicates coor-

dination in space and time ofcytoskeletal changes that affectthe formation of protrusions andadhesions at the migration frontbut also that elicit retraction of theold adhesions at the rear of the cell(43). The Rho family of GTPasesplays a key role in the regulation of



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migration-related processes, with Rac and Cdc42 drivingformation of the migration front and Rho controlling con-traction at the rear (36, 43). Filamins participate in responsesto extracellular signals that affect the cytoskeleton and causechanges in motility (12–14, 44). Our experiments showedthat endothelial cells with low levels of filamin B present aflattened phenotype with increased adhesion to extracellular

matrix (focal adhesions and con-tacts) in the basal state. After stim-ulation with VEGF, cells extendedprotrusions, polymerized actin,and generated stress fibers butlacked a well defined migrationfront. Altogether, these cells ap-peared to be defective in the stabi-lization of new adhesions by inter-action with the extracellular matrixor in detachment of the old adhe-sions. These defects could be ex-plained by an alteration of Vav-2-Rac-1-PAK signaling. In endothelialcells, PAK activity is necessary formigration, and it is up-regulated bythe formation of new adhesions; inaccordance with this, overexpres-sion of a PAK dominant negativeform inhibits cell migration (45).However, transfection of a consti-tutively active PAK mutant wasalso detrimental for migrationbecause PAK inactivation is re-quired for detachment and tailretraction. Taken together, theseresults indicate that a delicate bal-ance in the activity of PAK is nec-essary during cell migration (45).In our filamin B depletion experi-ments, we observe a decrease incell migration associated with anincrease of focal adhesions; thesechanges are accompanied by de-regulation of Rac-1-PAK activi-ties, which appear increased evenin basal conditions. Moreover,we have shown that alteration ofRac-1 normal activity in endothe-lial cells by overexpression of con-stitutively active or dominant neg-ative Rac-1 isoforms affects theendothelial phenotype and also

the ability of endothelial cells to migrate and organize incollagen gels. Therefore, it can be speculated that filamin Bdepletion causes a disruption of Rac-1-PAK signaling thatresults in PAK deregulation and defects in endothelial cellmigration. We also show that expression of filamin B isessential for translocation of Rac-1 to the plasma membrane

FIGURE 5. Filamin B depletion causes overstimulation of Rac-1 and PAK-4/5/6 basal activities in the absence of any effect on ERK1/2. A, Rac-1 activitywas measured using a G-LISA assay. Unrelated siRNA (UNr)- or siRNA-FLNB3-transfected cell lysates were incubated with an active Rac-binding protein boundto the bottom of a 96-well plate. After extensive washing, bound active Rac was detected with an anti-Rac antibody, followed by incubation with a secondaryantibody conjugated with horseradish peroxidase. Detection reagent was added, and absorbance was measured at 595 nm. Results are expressed as relativeto unrelated siRNA-transfected cells and represent an average of three experiments � S.E. B, HUVEC were transiently transfected with the different siRNAs asdescribed earlier. After 24 h of transfection, cells were depleted of growth factors for 16 h and stimulated with 20 ng/ml VEGF for the indicated periods of time.Cells were lysed, and FLNB, phospho-PAK-4/5/6 (pPAK-4/5/6), total PAK4, phospho-ERK1/2 (pERK), total ERK1/2, and �-actin (as a loading control) wereimmunodetected by Western blotting. A representative blot of five different experiments is shown. Densitometric quantification relative to loading controland mean � S.E. of the five experiments is shown. Phospho-PAK-4/5/6 was significantly increased by filamin B depletion (p � 0.05 t test).

FIGURE 6. Filamin B depletion blocks Rac-1 VEGF-induced translocation to plasma membrane. A, HUVECwere transiently transfected with an unrelated control siRNA (Unr) or siRNA-FLNB3 as described, in the absenceor presence of 20 ng/ml VEGF for 30 min. Cells were lysed, and FLNB, Rac-1, and tubulin (as a loading control)were immunodetected by Western blotting. A representative blot of three different experiments is shown.B, immunolocalization of F-actin (blue), Rac-1 (green), and filamin B (red) in HUVEC transiently transfected withan unrelated control siRNA or siRNA-FLNB3, as described, in the presence of 20 ng/ml VEGF for 30 min. Bar,40 �m.



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after VEGF stimulation, an essential step for cell migration(36–38). Thus, filamin B plays a key role in the control ofRac-1-PAK signaling necessary for the cycles of attachmentand detachment that take place during cell migration. Ourresults suggest that filamin B exerts this role by acting as ascaffolding protein, facilitating the interaction of Rac-1 andthe guanine nucleotide exchange factor Vav-2, which con-trols Rac-1 stimulation in endothelial cells (39). Filamin B isbound to Vav-2 and Rac-1 in basal conditions, ensuring theproper interaction of these proteins with VEGFR2 and

proangiogenic integrins after VEGFstimulation and their subsequentactivation. In the absence of fil-amin B, this signaling cascade isaltered; VEGFR2 can still stimu-late Src and ERK, but Vav-2 phos-phorylation is inhibited. In theseconditions, Rac-1 is deregulated,and its activity increases. Themechanism behind this increase inRac-1 activity in filamin B-de-pleted cells will require furtherinvestigation. We can speculatethat alteration of Rac-1 intracellu-lar location could affect its normalcycles of activation/inactivationnecessary for normal endothelialcell migration. An alternative sce-nario is that filamin B could facili-tate the interaction of Rac-1 notonly with the activator Vav-2 butalso with a GTPase-activatingprotein (GAP) and thus mediateits inactivation. A candidate GAPfor this role was FilGAP, a Rho-regulated GTPase-activating pro-tein that inactivates Rac-1, pro-moting cell retraction through itsinteraction with filamin A (46).We failed to detect interactionbetween FilGAP and filamin Bby immunoprecipitation experi-ments (data not shown), but thepossibility remains that filamin Binteracts with another GAP thatmight modulate Rac-1 activity.Our results suggest that the ef-

fects exerted by filamin B on endo-thelial cells are mediated by itscapacity to form signaling com-plexes, including FLNB, Rac-1,Vav-2, VEGFR2, and integrin �v�5,following VEGF stimulation. A sim-ilar scaffolding role has beendescribed for filamin A; this proteinhas been shown to interact withprostate-specific membrane anti-gen and stimulate Rac-1 activity

and endothelial cell migration through regulation of itsinteraction with integrins (47). Furthermore, as describedbefore, filamin A mediates the interaction between Rac-1and FilGAP (46). An article from Jeon et al. (41) has alreadydescribed an interaction between Rac-1 and filamin B, pro-posing a role for filamin B as a scaffold protein in type Iinterferon signaling, linking Rac-1 and the JNK cascademodule in HeLa cells. Our results confirm this interactionand describe a new function for filamin B in the endothelialcell context, not only in maintaining cellular structure but

FIGURE 7. Alteration of Rac-1 activity affects HUVEC phenotype and ability to form tubular structures.A, HUVEC cultured in two-dimensional (2D) plates were transiently transfected with GFP, GFP-Rac1-V12 (aconstitutively active form of Rac-1), or GFP-Rac1-M7 (a dominant negative form of Rac-1) as described. After24 h, phase-contrast and fluorescent images of each experiment were captured on a Leica inverted phase-contrast microscope (�20). Overexpression of GFP-Rac1-V12 resulted in a flat phenotype, whereas GFP-Rac1-M7 caused a mixed effect, with some cells presenting a flat phenotype and others showing a normalshape. B, after 24 h, HUVEC transfected as in A were trypsinized and immersed in collagen gels (three-dimen-sional cultures (3D)) in the presence of 40 ng/ml VEGF for an additional 24 h. Phase-contrast and fluorescentimages of each experiment were captured on a Leica inverted phase-contrast microscope (�20). Cells trans-fected with GFP were readily incorporated into pseudotubular structures formed in collagen gels (arrows),whereas cells with deregulated Rac-1 activity were excluded from these structures.



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also in facilitating cell migration following VEGF stimula-tion, an essential process for angiogenesis.

Acknowledgments—We thank Jacques Pouyssegur (Institute ofCellular Signaling, Developmental Biology, and Cancer Research,CNRS, Nice, France), Cristina Gamell and Gustavo Egea (Univer-sitat de Barcelona), Mireia Dunach and Jesus Ruberte (UniversitatAutonoma de Barcelona), Alan Hall (Memorial Sloan-KetteringCancer Center, New York), and David García Molleví (InstitutCatala d’Oncologia-IDIBELL) for reagents and technical support;Francesc Mitjans (Merck Farma y Química, Barcelona, Spain) forintegrin antibodies; and Esther Castano and Benjamín Torrejon(Serveis Cientificotecnics de la Universitat de Barcelona), EsterAdanero and Aurea Navarro (Universitat de Barcelona), andMaria Molas (Universitat Autonoma de Barcelona) for technicalsupport.

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Francesc Ventura and Francesc ViñalsFigueras, Sandor S. Shapiro, Toshiro Takafuta, Oriol Casanovas, Gabriel Capellà,

Beatriz del Valle-Pérez, Vanesa Gabriela Martínez, Cristina Lacasa-Salavert, AgnèsEndothelial Cell Motility through Its Interaction with Rac-1 and Vav-2

Filamin B Plays a Key Role in Vascular Endothelial Growth Factor-induced

doi: 10.1074/jbc.M109.062984 originally published online January 28, 20102010, 285:10748-10760.J. Biol. Chem.

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